The present invention relates to an improved method for mass spectrometric analysis, in particular for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), using nanoparticles, with an analyte being added to a nanoparticle suspension, and the suspension containing the bound analyte then being deposited directly on a MALDI sample carrier and investigated by mass spectrometry, and to nanoparticles suitable for this method.
Mass spectrometry is a method for elucidating the structure of substances, with atomic and molecular particles being separated according to their mass. It is based on a reaction between molecules and electrons or photons. Bombardment of the sample with electrons results, as a consequence of the elimination of electrons, in positive molecular ions which then dissociate into various ionic, free radical and/or neutral fragments. Molecular ions and fragments are separated in suitable separating systems according to the size of the mass number. Thus, mass spectrometry differs from real molecular spectrometric methods such as UV/vis, IR or NMR spectroscopy by using molecular ions and fragments resulting from chemical dissociation reactions as a consequence of an ionization process for elucidating the structure of substances.
The ions which are formed are separated according to their mass/charge (m/z) ratio in an analyzer, for example a magnetic or electric field. A mass spectrometer therefore generally consists of the following main components: the sample substance is vaporized in the inlet system and introduced in vapor form into the ion source in which the ionization takes place owing to, for example, electron impacts. The analyzer serves to separate, i.e., focus, the radical cations and cations formed in the ion source according to the mass-to-charge ratio. The substance vapor which has passed from the inlet system into the ion source is bombarded there by electrons which are emitted by an electrically heated metal wire, the filament. Between the filament and the electron target, the sample carrier, there is the so-called chamber voltage which accelerates the electrons to the desired energy.
Time-of-flight mass spectrometers have dynamic ion separation systems. In the time-of-flight mass spectrometer, ions differing in mass are separated on the basis of the differences in their time of flight for a predetermined path length. The accelerated ions enter the flight tube in which the end is reached faster by lighter than by heavier ions. Besides electron impact ionization, further ionization methods used are field ionization and field desorption ionization. In field ionization, positive ions are generated by removing an electron in a strongly electric field. Owing to the low energy of the molecular ions, only few fragmentations occur. To ionize compounds which are difficult to vaporize (field desorption ionization), a solution of these compounds is applied to an activating metal wire connected as anode. The ions resulting after application of the electric field are desorbed.
Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has developed in recent years into an important method for analyzing a wide variety of substances, especially proteins. The main advantages of this method include extremely rapid positive identification of an analyte, for example of a protein, through its mass-to-charge ratio (m/z) and the extremely low limit of detection, which is in the femtomole region or below.
Protein biochips have been developed in recent years for mass spectrometric analysis. In these, chemically functional groups are covalently tethered on the chip surface as self-assembled monolayers (SAM). Appropriately prepared proteins are attached as receptors to these systematically disposed tether molecules. Subsequently, libraries of optional binding partners are applied to the surface. Excess material is removed by using suitable cleaning steps, while specifically bound ligands remain tethered to the surface and can be analyzed directly by mass spectrometry.
There are, however, two problems in principle with the binding of tether proteins used to capture ligands on flat surfaces. A flat surface considerably restricts the amount of tether proteins which can be bound in a relatively small region, and the analyte is not efficiently aligned in relation to the solid-phase capture surface.
These problems are therefore solved by employing particulate binding matrices, since particles, especially particles in the nanometer range, have a very large surface area. Particulate systems with magnetic properties are employed in particular for high-affinity attachment, separation and preconcentration of proteins (Merchant and Weinberger, Electrophoreses, 21 (2000), 1164–1167). Sample preparation for MALDI-TOF MS analysis is common knowledge. For example, antibodies of a protein to be isolated are immobilized on the particle surface and then the corresponding protein is captured from complex matrices (Hurst et al., Anal. Chem., 71 (1999), 4727–4733). Particles which have been employed in particular are magnetic particles, polystyrene particles and Sephacryl particles. The molecules are coupled to the particles via a glutaraldehyde bridge or via a direct linkage via CNBr-activated carbohydrates.
However, it has emerged that the particulate systems employed to date are not entirely compatible with the actual MALDI analysis method and therefore must be removed before application to the sample carrier in order to avoid interference with the MALDI process. This means that the particulate binding matrices are used in order to isolate and purify analytes, but must be released again from the immobilized analytes in an additional operating step before the sample application to the MALDI sample carrier. Only then is it possible to analyze the analytes by means of the MALDI-TOF MS method.